The United States government has rights in this invention by virtue of grants from the National Institutes of Health, DK 16272 and GM 42504 and from the National Science Foundation, DMB-890538.
This application claims priority to Japanese patent application No. 4-81664 filed Mar. 2, 1992.
The present invention is an isolated nucleic acid sequence encoding the heme-regulated eukaryotic initiation factor 2.alpha., kinase, and methods of use thereof in inhibition of cellular proliferation.
Heme controls the synthesis of protein in reticulocytes. In heme-deficiency, there is diminished initiation of protein synthesis with disaggregation of polyribosomes. The principal mechanism of the inhibition of initiation of protein synthesis is the phosphorylation of the .alpha.-subunit of the eukaryotic initiation factor 2, eIF-2e. In addition to heme-deficiency, oxidized glutathione (GSSG) and low levels of double stranded RNA inhibit initiation by promoting phosphorylation of eIF-2.alpha..
The translation of mRNA in eukaryotic cells occurs in the cytoplasm. In the first step of initiation, free 80 S ribosomes are in equilibrium with their 40 S and 60 S subunits. In the presence of eIF-3, 40 S subunits bind the eIF-3and eIF-4C to form a 43 S ribosomal complex; the binding of eIF-3 and eIF-4C to the 40 S subunit inhibits the joining of the 60 S subunit.
In the next step, eIF-2 binds GTP and the initiator tRNA, Met-tRNA.sub.f, in a ternary complex. The binding by eIF-2 is specific for both guanine nucleotides and for Met-tRNA.sub.f. The ternary complex now binds to the 43 S ribosomal complex to form the 43 S preinitiation complex. The 43 S preinitiation complex binds mRNA in an ATP-dependent reaction in which eIF-4A, eIF-4B, and eIF-4F form a complex with the mRNA. The product of the binding of mRNA to the 43 S structure is bound close to the ribosome and the AUG initiator codon is downstream from the cap structure.
The joining of the 48 S preinitiation complex and the 60 S subunit is catalyzed by eIF-5 which has a ribosome-dependent GTPase activity. The joining reaction is accompanied by the release of the initiation factors eIF-3 and eIF-4C, eIF-2 is translocated to 60 S subunit as a binary complex, eIF2-GDP. The product of the joining reaction is the 80 S initiation complex. Formation of the active 80 S initiation complex is the final step in initiation. The Met-tRNA.sub.f is positioned in the P (peptidyl) site on the ribosome for the start of polypeptide elongation.
The sequence of steps in the process of initiation affords several opportunities for regulation. These include the recycling of eIF-2 after its release as the eIF-2-GDP complex; the formation of the ternary complex; and the relative affinities of mRNAs for eIF-2 and for eIF-4A, -4B, and -4F in determining the relative rates of translation of the mRNAs.
A schematic summary of eukaryotic initiation is shown in FIG. 1. Heme-deficiency inhibited initiation of protein synthesis is characterized by a brief period of control linear synthesis, followed by an abrupt decline in this rate and by disaggregation of polyribosomes, associated with a decrease in the formation of the eIF-2-Met-tRNA.sub.f -GTP ternary complex and the 40 S-eIF-2Met-tRNA.sub.f -GTP 43 S initiation complex. The fundamental mechanism for the inhibition is the activation of cAMP independent protein kinases that specifically phosphorylate the 38-kDa .alpha.-subunit of eIF-2 (eIF-2.alpha.). Dephosphorylation of eIF-2.alpha. accompanies the recovery of protein synthesis upon addition of hemin to inhibited heme-deficient lysates.
The heme-regulated eukaryotic initiation factor 2.alpha. (eIF-2.alpha.) kinase, also called heme-regulated inhibitor (HRI), plays a major role in this process. HRI is a cAMP-independent protein kinase that specifically phosphorylates the e subunit (eIF-2.alpha.) of the eukaryotic initiation factor 2 (eIF-2). Phosphorylation of eIF-2.alpha. in reticulocyte lysates results in the binding and sequestration of reversing factor RF, also designated as guanine nucleotide exchange factor or eIF-2B, in a RF-eIF-2(.alpha.P) complex; the unavailability of RF, which is required for the exchange of GTP for GDP in the recycling of eIF-2 and in the formation of the eIF-2-Met-tRNA.sub.f -GTP ternary complex, resulting in the cessation of the initiation of protein synthesis.
Although the mechanism of regulation of protein synthesis by HRI has been extensively studied, little is known about the structure and regulation of HRI itself. Chen, J.-J., et al., Proc. Natl. Acad. Sci., USA 88:315-319 (1991) previously reported the amino acid sequences of three tryptic peptides of heme-reversible HRI. HRI peptide P-52 contains the sequence Asp-Phe-Gly, which is the most highly conserved short stretch in conserved domain VII of protein kinases as presented by Hanks, Quinn, and Hunter, Science 241:42-52 (1988). The N-terminal 14 amino acids of HRI peptide P-74 show 50-60% identity to the conserved domain IX of kinase-related transforming proteins. These findings are consistent with the autokinase and eIF-2.alpha. kinase activities of HRI. As reported by Pal et al., Biochem. 30:2555-2562 (1991), this protein appears to be erythroid-specific and antigenically different in different species.
In view of the activity and relationships of HRI to other protein kinases involved in cellular transformation, it would be advantageous to have the nucleic acid sequence encoding HRI. However, since the gene is only expressed during a very limited time period, i.e., during erythroid differentiation, and in an extremely minuscule amount, this was not a simple process. Moreover, even though three peptides isolated by tryptic digest had been sequenced, it was not clear if these were from HRI or from a contaminant of the HRI preparation. Obtaining a library containing a full length HRI cDNA is also difficult.
It is therefore an object of the present invention to provide a cDNA sequence encoding HRI.
It is a further object of the present invention to provide methods for expression of HRI in mammalian cells.
It is still another object of the present invention to provide methods of use of the isolated DNA sequence encoding HRI to inhibit cell proliferation, by inhibiting protein synthesis, especially of transformed cells and in diseases such as psoriasis.
It is another object of the present invention to provide methods of use of the sequence encoding HRI and dsI to induce cellular differentiation and treat cancers involving arrested differentiation.